Project Summary/Abstract Approximately 25% of acute myeloid leukemias (AML) contain an internal tandem duplication (ITD) in the juxtamembranal region of FLT3. Even with standard chemotherapies, FLT3-ITD AML is associated with a five- year survival rate of only 15% as compared to 40% for wild-type FLT3 AML. Therefore, FLT3 inhibitors have been developed as a targeted treatment approach. Although the inhibitors show considerable efficacy in vitro and in animal models, clinical trials using FLT3 inhibitors as single agents have been underwhelming, with few complete remissions and a high rate of relapse and resistance. Thus, there is a need for a FLT3 inhibitor that increases complete remission rates and reduces relapse of FLT3-ITD AML patients. To combat the many mechanisms of resistance used by cancers, polypharmacology is gaining popularity in the field of drug discovery. This method, using a single agent to interact with multiple targets, has several advantages over selective inhibitors or drug combinations; primarily, targeting multiple pathways simultaneously decreases the likelihood of resistant clones. We propose that Interleukin-1 Receptor Associated Kinase 1 (IRAK1) and IRAK4 are candidates for targeted therapy in combination with FLT3 inhibition. The IRAK1/4 kinase complex mediates signaling downstream of the IL-1 (IL1R) and Toll-like (TLR) Receptors; however, these signaling pathways are also implicated in several hematopoietic malignancies, and the efficacy of IRAK1/4 inhibition has been demonstrated in preclinical models of AML, Myelodysplastic Syndrome, and Acute Lymphoid Leukemia. In preliminary data, treatment of FLT3-ITD positive AML cells with the second generation FLT3 inhibitor, AC220, results in a marked increase in IRAK1/4 phosphorylation, an indication of IRAK1 and IRAK4 activation. Collectively, these findings suggest that IRAK1/4 activation permits outgrowth of AC220-resistant cells and is a suitable candidate to target in FLT3-ITD positive AML. Our central hypothesis is that the inhibition of FLT3-ITD results in a compensatory increase in IRAK1/4 activity to promote cell survival; thus, targeting both pathways simultaneously will decrease the likelihood of the development of resistant clones. To test this hypothesis, we propose the following aims: (1) Determine the crosstalk between FLT3 and adaptive IRAK1/4 signaling in AML and (2) Determine the efficacy of a novel polypharmacologic IRAK1/4 and FLT3 inhibitor. Collectively, these studies will dissect adaptive FLT3-ITD signaling in AML, elucidate a novel resistance pathway in AML, and provide a novel and improved therapeutic strategy to treat FLT3-ITD AML.